WO1997034012A1 - Physiologically active substances tkr1785's, process for the preparation thereof, and microbe - Google Patents

Physiologically active substances tkr1785's, process for the preparation thereof, and microbe Download PDF

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Publication number
WO1997034012A1
WO1997034012A1 PCT/JP1997/000770 JP9700770W WO9734012A1 WO 1997034012 A1 WO1997034012 A1 WO 1997034012A1 JP 9700770 W JP9700770 W JP 9700770W WO 9734012 A1 WO9734012 A1 WO 9734012A1
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Prior art keywords
tkr1785
physiologically active
culture
active substance
tkr
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PCT/JP1997/000770
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French (fr)
Japanese (ja)
Inventor
Kazutoh Takesako
Naoyuki Awadu
Yoshie Yoshikawa
Eiko Koyama
Ikunoshin Kato
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Takara Shuzo Co., Ltd.
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Application filed by Takara Shuzo Co., Ltd. filed Critical Takara Shuzo Co., Ltd.
Priority to AU19396/97A priority Critical patent/AU1939697A/en
Priority to US09/142,689 priority patent/US6103767A/en
Priority to EP97907278A priority patent/EP0897988B1/en
Priority to JP53243897A priority patent/JP3273948B2/en
Priority to DE69733016T priority patent/DE69733016T2/en
Publication of WO1997034012A1 publication Critical patent/WO1997034012A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C237/12—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by carboxyl groups
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the present invention provides a biologically active substance TKR 1785 useful as a therapeutic agent for fungal infections, allergic diseases and immunity, and a method for producing the same, and further produces a biologically active substance TKR 1785 Microorganisms.
  • Fungi are known to infect humans, animals, plants and the like and cause various diseases. For example, it causes superficial mycosis in human skin, oral cavity, etc., systemic mycosis in internal organs, brain, etc., and similar infections in animals such as pets and livestock. Wake up. Furthermore, it causes various diseases on plants such as fruit trees and vegetables.
  • Candida which infects the skin, oral cavity, vagina, etc.
  • Trichophyton cause of athlete's foot
  • Malassezia cause of wind
  • allergic diseases such as asthma, atopic dermatitis, and allergic rhinitis have been rapidly increasing. Most of allergic diseases are sensitized to an antigen causing the disease, and an IgE antibody (reagin antibody) specific to the antigen allergen is produced in serum and tissue. again Binds to mast cells or basophils when exposed to the allergen
  • Chemical mediators include those already contained in mast cells and eosinophil granules, which are released out of cells by degranulation upon activation, such as histamine, serotonin, and Some of them are newly synthesized by activation of mast cells and others, such as eosinophil factors.
  • Systemic effects are the result of an IgE-basophil response to antigens in blood vessels.
  • allergic diseases in which various substances in the environment, such as mites and various pollens, and antigenic substances contained in foods, etc., act as allergens.
  • allergens there are many allergic diseases caused by fungi, and allergens derived from Candida, Aspergillus, Alternaria, Cladosporium, Malassezia, Penicillium, etc. work as a cause.
  • antifungal agents Only a very small number of antifungal agents are currently known that can be used to treat or defend against such fungal infections and contamination-especially humans.
  • Other therapeutic agents for systemic infections in animals include amphotericin B, flucitrosin, and miconazo. And fluconazole. These compounds have problems in efficacy, toxicity, antibacterial spectrum, etc., and have not been sufficient as therapeutic agents.
  • An object of the present invention is to provide a novel bioactive substance useful as a therapeutic agent for fungal infectious diseases, allergic diseases, and immunity in view of the above-mentioned situation.
  • we isolated many microorganisms from nature isolated the biologically active substances produced by them, and examined their biological properties.
  • the culture of microorganisms belonging to the genus Penici Ilium It was found that a biologically active substance showing antibacterial activity against pathogenic fungi such as Candida, Aspergillus, Clipcoccus and Malassezia was present in the product. Subsequently, the present inventors isolated these physiologically active substances and examined their physicochemical properties.
  • TKR 1785 (hereinafter referred to as “TKR 1785—1”) and TKR 1785-II (hereinafter collectively referred to as “TKR 1785 class”).
  • TKR 1785-classes are involved in allergic reactions
  • the present inventors have found that the enzyme has a physiological activity on the immune system, including the inhibitory activity of the enzyme, and have completed the present invention.
  • the first present invention provides the following general formula (A):
  • R is one CH (CH 3 ) 2 or —CH (CHa) C 2 H s ).
  • a second aspect of the present invention is to culture a strain belonging to Penicillium (Penici Ilium), which produces a physiologically active substance TKR1785, and thereafter, from the culture of the strain, This is a method for producing a physiologically active substance TKR1785 for isolating a target substance.
  • the third invention is a microorganism which belongs to the genus Beninium (Penic11ium) and produces the physiologically active substances TKR1785.
  • the fourth invention is a pharmaceutical composition containing the physiologically active substance TKR1785.
  • FIG. 1 is a diagram showing an ultraviolet absorption spectrum of the physiologically active substance TKR1785-1.
  • the vertical axis indicates the wavelength (nm).
  • FIG. 2 is a diagram showing an infrared absorption spectrum of the physiologically active substance TKR1785-1.
  • the horizontal axis shows the wave number (cm_ ').
  • FIG. 3 is a diagram showing a ' ⁇ -NMR spectrum of TKR1785-I, a biologically active substance.
  • the horizontal axis shows the chemical shift value (ppm).
  • Figure 4 is a diagram showing a physiologically active substance TKR 1 7 8 5- 1 '3 G 1 ⁇ 1 ⁇ 13 ⁇ 4 spectrum.
  • the horizontal axis shows the chemical shift value (P pm).
  • FIG. 5 is a diagram showing the elution position of the physiologically active substance TKR1785-I in HPLC.
  • the vertical axis indicates the retention time (minutes), and the horizontal axis indicates the relative ultraviolet absorption intensity.
  • FIG. 6 is a diagram showing an 'H-NMR spectrum of the physiologically active substance TKR1785-II.
  • the horizontal axis shows the chemical shift value (P Pm).
  • Figure 7 is a biologically active substance TKR 1 7 8 5 - a diagram showing the 1 1 of 13 ⁇ over 1 ⁇ 13 ⁇ 4-spectrum Le. The horizontal axis shows the chemical shift value (P pm).
  • FIG. 8 is a diagram showing the elution position of the physiologically active substance TKR1785-II in HPLC.
  • the vertical axis indicates the retention time (minutes), and the horizontal axis indicates the relative ultraviolet absorption intensity.
  • the physiologically active substance TKR1785-I has the following physicochemical properties (1), (2), (3), (4), (5), (6) and (7).
  • Mass spectrum by FAB-MS method has a peak of mZz518 [M + H].
  • the main absorption wave numbers of the infrared absorption spectrum by the KBr method are 3410 cm- ', 2920 cm-', 2850 cm- ', 1670 cm- ', 1540 cm—', 1470 cm— ', 1 210 cm "', 1140 cm ', 1500 cm I, 8400 cm—', 800 cm 720 cm- 1 .
  • TKR1785-I had the chemical structure shown in formula (I).
  • the physiologically active substance TKR1785-II has the following physicochemical properties (8), (9), (10) and (11).
  • the TKR1785 can be produced by culturing a strain that produces the TKR1785 in the genus Penicillium, and then isolating the strain from a culture of the strain. it can.
  • the strain used in the present invention is not particularly limited as long as it belongs to Penicillium and produces the above-mentioned TKR1785s.
  • Penicillium sp. TKR 175 strains (hereinafter referred to as "TKR 175 strains").
  • the above-mentioned TKR1785 strain is a new strain not described in the literature, and was isolated and identified by the present inventors for the first time from a sample collected in Hyogo Prefecture. Has the property of advantageously producing Hereinafter, the mycological properties of the TKR1785 strain will be described in detail.
  • Table 1 shows the color tone of the colonies (hereinafter, also referred to as J colonies j) of the above TKR1785 strains in various media.
  • the color tone in the table is based on the color name according to Japanese Industrial Standard JISZ 8102 (1989), after inoculating the medium, culturing at 25, and observing after 7 It is shown.
  • J colonies j the colonies of the above TKR1785 strains in various media.
  • J colonies j the color tone in the table is based on the color name according to Japanese Industrial Standard JISZ 8102 (1989), after inoculating the medium, culturing at 25, and observing after 7 It is shown.
  • the TKR1785 strain grows rapidly on a malt extract agar medium, potato dextroth agar medium, Wapec agar medium, and the like, and the surface of the colony is in the form of a belode, and the center is slightly raised.
  • the conidiophores of the TKR 1785 strain have a smooth surface of 90 to 270 x 8 to 3.0 m, and form almost bicyclic biosymmetric Benicili.
  • the metres are 12.0 to 14.0 2.8 to 3.2 2, and 2 to 4 trees are clustered, and the fireride is 9.0 to 10.0 1.8 to 2.4. It becomes a ring shape in im.
  • Conidia globose or et subglobose the smooth surface, the size of 2. 2 ⁇ 3. 2 x 2. 4 ⁇ 4.
  • mycological properties of c the TKR 1 7 8 5 strains is 0 m, Physiological properties are as shown below.
  • the temperature range where growth is possible is 10-30, and the optimum growth temperature The temperature is around 25 ° C.
  • Growth pH range The range of pH that can be grown is pH 3 to pH 9, and the growth optimal pH is around pH 5.
  • a bacterial species having the above-mentioned mycological properties is referred to as Carlos' Ramirez (Car 10 s).
  • the above TKR1785 strain can be identified as a strain belonging to Benicillium by searching for strains of the genus Penicillium described in the literature such as 1982.
  • TKR 1785 strain in addition to the above-mentioned TKR 1785 strain, natural or artificial mutant strains of the TKR 1785 strain, other strains belonging to the genus Penicillium, etc. Microorganisms having the ability to produce such species can be used.
  • the productivity of the above-mentioned TKR1785 class of the above-mentioned microorganisms is the same as that of Fig. 5 for physiologically active substances contained in an appropriate culture of a strain of Nissan strain.
  • the KR1785s are produced by inoculating a strain producing the TKR185s into a nutrient-containing medium and culturing the same.
  • the carbon source include glucose, fructos, saccharose, starch, dextrin, glycerin, molasses, syrup, oils and fats, Aritsuki acid and the like.
  • nitrogen sources include, for example, soybean flour, cottonseed flour, corn brik, casein, peptone, yeast extract, meat extract, germ, urea, amino acid, ammonium salt, etc.
  • Organic nitrogen compounds, inorganic nitrogen compounds and the like can be mentioned.
  • examples of the salt include inorganic salts such as sodium salt, potassium salt, calcium salt, magnesium salt, and phosphate. Each of these may be used alone or in combination as appropriate.
  • the nutrient-containing medium may further contain, if necessary, heavy metals such as iron salts, copper salts, zinc salts and cobalt salts; vitamins such as biotin and vitamin B; Organic substances, inorganic substances, and the like that promote the production of TKR1785 can be added as appropriate.
  • heavy metals such as iron salts, copper salts, zinc salts and cobalt salts
  • vitamins such as biotin and vitamin B
  • Organic substances, inorganic substances, and the like that promote the production of TKR1785 can be added as appropriate.
  • an antifoaming agent such as silicone oil and polyalkylene glycol ether, a surfactant, and the like can be added to the nutrient source-containing medium, if necessary.
  • a method generally used for producing a physiologically active substance by culturing a microorganism can be employed, but a liquid culture method, in particular, a shaking or deep aeration stirring culture method is preferable. Can be used.
  • the above culture is preferably performed at 15 to 25, and the pH of the medium is usually pH 3 to 8, but is preferably around pH 5. In general, a sufficient production amount can be obtained in a culture period of 3 to 11 days.
  • TKR1785 is contained in the culture solution and the cells and accumulates in the culture.
  • the TKR1785 accumulated in the culture is separated from the culture using the physicochemical and biological properties of these physiologically active substances, and then, if necessary, It can be further purified and obtained.
  • the above separation can be carried out by extracting the whole culture with a non-hydrophilic organic solvent such as ethyl acetate, butyl acetate, black form, butanol, and methyl isobutyl ketone. Further, after the culture is separated into a culture solution and cells by filtration or centrifugation, the culture can be separated from each of the culture solution and the cells.
  • a non-hydrophilic organic solvent such as ethyl acetate, butyl acetate, black form, butanol, and methyl isobutyl ketone.
  • TKR1785 In order to separate TKR1785 from the culture solution, a method of extracting with the above-mentioned non-hydrophilic organic solvent can be adopted.Also, the culture solution is brought into contact with an adsorptive carrier, and Alternatively, a method may be employed in which TKR1785 is adsorbed on a carrier and then eluted with a solvent.
  • the carrier include activated carbon, powdered cellulose, and adsorbent resin.
  • the solvent may be used alone or in combination of two or more depending on the type and properties of the carrier.For example, an aqueous solution of a water-soluble organic solvent such as hydrated acetate or hydrated alcohol may be used. And the like in combination.
  • TKR1785 In order to separate TKR1785 from the above cells, a method of extraction with a hydrophilic organic solvent such as acetone can be adopted.
  • the crude extract of TKR'1785s thus separated from the culture can be subjected to a step of further purification, if necessary.
  • the above-mentioned purification can be carried out by a method usually used for separation and purification of a fat-soluble physiologically active substance. Examples of such a method include a method using a carrier such as silica gel, activated alumina, activated carbon, and an adsorbent resin.
  • high-performance liquid chromatography When column chromatography using silylation gel as a carrier is used, examples of the eluting solvent include chromatographic form, ethyl acetate, methanol, acetone, water, and the like. Can be used in combination.
  • examples of the carrier include chemically bonded silica gel having an octadecyl group, an octyl group, and a phenyl group bonded thereto; a polystyrene-based porous polymer gel;
  • the mobile phase for example, an aqueous solution of a water-soluble organic solvent such as hydrated methanol and hydrated acetonitrile can be used.
  • the TKR1785s of the present invention can be used in medicine as it is or as a pharmacologically acceptable salt thereof.
  • the pharmaceutical composition containing the above-mentioned TKR 17885 or a pharmacologically acceptable salt thereof is not particularly limited, and examples thereof include antifungal agents, antiallergic agents, and immunomodulators. Can be. In the present invention, it is preferable to use as an antiallergic agent, an immunomodulator, etc., but the use is not limited to these.
  • the present invention includes all pharmaceutical compositions containing 875 or a reasonably acceptable salt thereof.
  • the salt is not particularly limited as long as it is pharmacologically acceptable, and examples thereof include minerals such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrofluoric acid, and hydrobromic acid.
  • Salts of acids formic acid, acetic acid, tartaric acid, lactic acid, citric acid, fumaric acid, maleic acid, succinic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalenesulfonic acid, carboxylic acid Salts of organic acids such as dimethyl sulfonate; salts of alkali metals such as sodium, potassium and calcium, and salts of alkaline earth metals.
  • the TKR1785 class of the present invention or a pharmacologically acceptable salt thereof is used.
  • the carrier examples include solid, semi-solid or liquid diluents, fillers, and other auxiliaries for formulation. One or more of these can be used.
  • the above pharmaceutical composition is preferably administered in the form of a dosage unit, and can be administered orally, intraosseously, topically (such as transdermally) or rectally. Naturally, the above pharmaceutical composition is administered in a dosage form suitable for these administration methods.
  • the dose as an antifungal agent, an antiallergic agent or an immunomodulator is determined by the age, body weight, etc. It is desirable to make adjustments in consideration of the condition of the patient, the administration route, the nature and extent of the disease, etc., but usually, for humans.
  • the TKR1775-class of the present invention, or The amount of the active ingredient of the pharmacologically acceptable salt is in the range of 10 to 2000 mg per day. Dosages below the above range may be sufficient, while conversely, doses above the above range may be required. When administering large amounts, take several times a day. It is advisable to split the dose.
  • a powder can be produced by appropriately preparing the TKR1785 class of the present invention or a pharmacologically acceptable salt thereof.
  • the powder may be a finely-divided TKR1775-class of the present invention or a pharmaceutically acceptable salt thereof, and then a finely divided pharmaceutical carrier, for example, edible carbohydrates such as starch and mannitol. It is manufactured by mixing with others. If desired, flavoring agents, preservatives, dispersing agents, coloring agents, flavors and the like may be mixed.
  • Parenteral administration can be accomplished, for example, by using a liquid dosage unit form for subcutaneous, intramuscular or intravenous injection, such as a solution or a bumper.
  • a liquid dosage unit form for subcutaneous, intramuscular or intravenous injection such as a solution or a bumper.
  • These compounds can be used to convert a certain amount of TKR1785 of the present invention or a pharmacologically acceptable salt thereof into, for example, an aqueous or oily vehicle suitable for injection purposes. It is manufactured by suspending or dissolving in a liquid carrier, and then sterilizing the suspension or solution.
  • the above-mentioned topical administration can be carried out, for example, by using external preparations such as liquids, creams, powders, bases, gels, and ointments.
  • external preparations such as liquids, creams, powders, bases, gels, and ointments.
  • These compounds can be used to convert a certain amount of TKR1785 of the present invention or a pharmaceutically acceptable salt thereof into fragrances, coloring agents, fillers, surfactants suitable for the purpose of external preparations , moisturizers, emollients, gelling agents, carriers, save agent, c rectal administration is prepared by combining one or more of such stabilizers, TKR 1 7 8 5 such present invention, or, An aliquot of the pharmacologically acceptable salt is mixed with low-melting solids such as higher esters such as myristyl palmitate, polyethylene glycol, cocoa butter, and mixtures thereof. It can be performed using an agent or the like.
  • Example 1 Example 1
  • the culture solution thus obtained was centrifuged and separated into a supernatant and cells.
  • 1 L of methanol was added, mixed well, and extracted, and then concentrated under reduced pressure.
  • the pH was adjusted to 2.
  • 300 ml of ethyl acetate was added and mixed well, followed by washing with ethyl acetate.
  • the pH of this aqueous layer was adjusted to 9, and 300 ml of ethyl acetate was added to perform extraction.
  • the extract was concentrated under reduced pressure to obtain 52 mg of residual brew.
  • the obtained residue was dissolved in 0.4 ml of methanol and subjected to high performance liquid chromatography to obtain two antifungal active fractions I and II.
  • the fractions were concentrated under reduced pressure to obtain 16 mg and 3 mg of purified TKR1785-I and TKR1785-II, as white powders.
  • the conditions for high-speed liquid mouth chromatography were as follows.
  • a JMS-DX302 mass spectrometer (manufactured by JEOL Ltd.) was used for mass spectrometry.
  • 'H-NMR spectrum in heavy dimethyl sulfoxide, standard substance: heavy dimethyl sulfoxide
  • 13C- NMR spectrum in heavy dimethyl sulfoxide, standard substance: heavy dimethyl sulfoxide
  • UV absorption spectrum analysis in methanol
  • a UV-250 type recording spectrophotometer manufactured by Shimadzu Corporation
  • TKR 1785-1 For infrared absorption spectrum analysis (KBr method), a 270-30 type infrared spectrophotometer (manufactured by Hitachi, Ltd.) was used. For amino acid analysis, L-850 type (manufactured by Hitachi, Ltd.) was used. The physicochemical properties of TKR 1785-1 are described below.
  • the purified white powder obtained by subjecting the obtained active fraction I to a high-pressure liquid chromatography and reducing the pressure under reduced pressure was analyzed by FAB-MS by mass spectrometry to obtain mZz 518 [M + H] + .
  • mZz 518 [M + H] + According to ' ⁇ -NMR spectrum measurement and 3 C-NMR spectrum measurement of this substance and analysis thereof, it was found to have 27 carbon atoms and 3 nitrogen atoms.
  • the 1 H-NMR spectrum is shown in FIG. 3, and the: 3 C-NMR spectrum is shown in FIG.
  • the ultraviolet absorption spectrum of this substance in methanol showed terminal absorption as shown in FIG.
  • the infrared spectrum measurement result of this substance by the KBr method was as follows.
  • Figure 2 shows the infrared absorption spectrum.
  • TKR1785-I has the chemical structure of the formula (I) It became clear that it had.
  • TKR1785-I was subjected to analysis by reversed-phase partition high-performance liquid chromatography (HPLC) using an LC-10A high-performance liquid chromatograph (manufactured by Shimadzu Corporation).
  • HPLC reversed-phase partition high-performance liquid chromatography
  • LC-10A high-performance liquid chromatograph manufactured by Shimadzu Corporation.
  • the conditions for high performance liquid chromatography were as follows.
  • TKR1785-I was eluted at the position shown in FIG.
  • physicochemical properties of TKR 1785-11-1 are described.
  • TKR1785-II was subjected to analysis by HPLC using an LC-10 ⁇ type high-performance liquid chromatograph (manufactured by Shimadzu Corporation). The conditions for high performance liquid mouth chromatography were the same as in the above analysis of TKR1785-1. As a result of the analysis, it was revealed that the above TKR1785-II was eluted at the position shown in FIG. Biological properties
  • the antibacterial spectrum against various microorganisms was examined using the obtained TKR1785.
  • the concentration was determined as the minimum growth inhibition level (gZm 1) by the liquid medium dilution method, at which the growth of the bacteria was almost completely inhibited.
  • Table 2 shows the results.
  • the minimum concentration that partially inhibits bacterial growth is The degree (/ gZm 1) was obtained and is shown in parentheses in Table 2.
  • YNBG indicates a medium containing 0.67% of yeast tonite trogen base (manufactured by Difco) and 1% of glucose.
  • BHI stands for a medium containing 0.5% of Brainhard Toin Hydrange Bouillon (manufactured by Mizusui Pharmaceutical Co., Ltd.): YN BG—Tween is 0.67% of yeast nutrient base, Represents a medium containing 0.5% of ton, 2% of glucose and 40.1% of tween. Table 2
  • the physiologically active substances TKR1785 of the present invention include Candida albicans, Candida 'Kefir', Cryptococcus 'Neoholmans, Malassezia' and Farfer. Antibacterial activity against pathogenic fungi was found to have. (2) Various enzyme inhibitory activities
  • TKR 1 785-I enzymes involved in the immune system, phospholipase A 2 (derived from swine spleen) and leukotriene (LT) synthase
  • TKR 1 7 8 5 - [inhibited phospholipase A 2 and LTC ⁇ synthase, an enzyme involved in ⁇ Rerugi first reaction.
  • MLR Mixed lymphocyte reaction
  • the spleen was excised from the C57BLZ6 mouse and BALB / c mouse, and homogenized in the medium to obtain a cell suspension.
  • a cell suspension derived from C57B LZ6 was applied to a nylon wool column to prepare a T cell-rich cell (responder cells).
  • a cell suspension derived from BALB / c was irradiated with X-rays to prepare stimulator cells.
  • Responder cells and stimulator cells are mixed at a 1: 1 ratio, cultured in a CO 2 .incubator for 4 days, H-thymidine is added, and after overnight culture, the cells are collected. And 3 H-thymidine incorporation were measured.
  • TK R 1785-1 shows a concentration-dependent] VILR inhibitory activity, and its 50% inhibitory activity was 0.41 / ig / m1.
  • TKR1785-I and TKR1785-II were intraperitoneally administered to ICR mice at 50 mg / kg, respectively.
  • the tablets are made by wet granulation using a 8% (w / w) aqueous solution of hydroxyquinpropylmethylcellulose as a binder. Condyles were produced. After mixing with magnesium stearate, it was formed into a tablet with a diameter of 7 mm and a weight of 130 mg per tablet using a tableting machine.
  • Formulation Example 2
  • TKR 1785-I was kneaded sufficiently with a small amount of water-absorbing blue, and then the remaining water-absorbing soft blue was gradually added and kneaded until uniform to produce descendants. The resulting sound was applied to the affected area 4 to 5 times a day.
  • TKR1785 useful as a clinical drug such as a therapeutic agent for fungal infections, allergic diseases and immunity, and the production thereof A method can be provided.

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Abstract

Novel physiologically active substances, TKR1785's useful as remedies for fungal infections or the like, and represented by general formula (A) wherein R is -CH(CH3)2 or -CH(CH3)C2H5.

Description

明細書 生理活性物質 TKR 1 7 8 5類、 製造方法及び微生物 技術分野  Description Physiologically Active Substances TKR 1785, Production Method and Microorganism
本発明は、 真菌感染症、 アレルギー性疾患及び免疫の治療剤として有 用な生理活性物質 TKR 1 7 8 5類、 及び、 それらの製造方法、 更に、 生理活性物質 TKR 1 7 8 5類を産生する微生物に関する。 背景技術  The present invention provides a biologically active substance TKR 1785 useful as a therapeutic agent for fungal infections, allergic diseases and immunity, and a method for producing the same, and further produces a biologically active substance TKR 1785 Microorganisms. Background art
真菌は、 ヒ ト、 動物、 植物等に感染して種々の疾病を引き起こすこと が知られている。 例えば、 ヒ トの皮庸、 口腔等に表在性真菌症を起こし、 内臓、 脳等に全身性真菌症を起し、 ぺッ 卜、 家畜等の動物に対しても同 様の感染症を起こす。 更に、 果樹、 野菜等の植物に対しても、 種々の病 害を起こす。  Fungi are known to infect humans, animals, plants and the like and cause various diseases. For example, it causes superficial mycosis in human skin, oral cavity, etc., systemic mycosis in internal organs, brain, etc., and similar infections in animals such as pets and livestock. Wake up. Furthermore, it causes various diseases on plants such as fruit trees and vegetables.
このうち、 ヒ 卜に感染して、 全身性真菌症を起こす原因真菌の主なも のとしては、 カ ンジダ （Candida ) . ク リプ トコ ッカス （Cryptococcus) ァスペルギルス (Aspergillus ) 等が知られ、 表在性真菌症では、 皮膚、 口腔、 膣等に感染するカンジダ、 手足の皮膚等に感染する白癬菌 （水虫 の原因） やマラセチア (Malassezia) (癱風の原因） 等が主なものと考 えられている。 生活環境中にはこれら以外にも多様な真菌が存在し、 動 植物の汚染を引き起こす原因と考えられている。  Of these, Candida, Cryptococcus, Aspergillus, and the like are known as the main fungi that cause systemic mycosis in humans. It is considered that the main causes of mycosis are Candida, which infects the skin, oral cavity, vagina, etc., Trichophyton (cause of athlete's foot) and Malassezia (cause of wind), which infect the skin of limbs. ing. Various other fungi exist in the living environment and are thought to cause animal and plant contamination.
近年、 喘息、 ア トピー性皮膚炎、 アレルギー性鼻炎を始めとするァ レ ルギー性疾患が急激に増大しつつある。 アレルギー性疾患の多く は、 そ の疾患の原因抗原に感作されることにより、 血清及び組織で抗原である ァレルゲンに特異的な I g E抗体 （レアギン抗体） が産生される。 再び そのアレルゲンに暴露された際に、 肥満細胞又は好塩基球と結合したIn recent years, allergic diseases such as asthma, atopic dermatitis, and allergic rhinitis have been rapidly increasing. Most of allergic diseases are sensitized to an antigen causing the disease, and an IgE antibody (reagin antibody) specific to the antigen allergen is produced in serum and tissue. again Binds to mast cells or basophils when exposed to the allergen
I g Eと特異的なアレルゲンが組み合わさり、 I g Eが細胞表面上で架 橋し、 I g E —抗原相互作用の生理学的効果を生じる。 これらの生理学 的効果には、 ケミカルメディエーターと呼ばれるものが関与している。 ケミカルメデイエ一ターとしては、 すでに肥満細胞、 好酸球の顆粒中 に含まれていたものが、 活性化による脱顆粒によって細胞外へ放出され るもの、 例えば、 ヒスタ ミ ン、 セロ トニン、 好酸球因子等と、 肥満細胞 の活性化によって新たに合成されるものがある。 後者の場合、 ホスホリ パーゼ A 2 の活性化に続き、 リポキシゲナーゼ、 シクロォキシゲナーゼ が作動し、 これらが膜りん脂質のァラキ ドン酸に作用し、 各種ロイコ ト リエン類、 トロンボキサン類等が生成される。 これらのケミカルメディ エーターは、 長期間の気管支平滑筋の収縮、 粘膜浮腫等のアレルギー性 炎症を起こす。 これらの効果は、 抗原が体内に入る経路及び肥満細胞又 は好塩基球上に I g Eが沈着するパターンに依存して、 全身的又は局所 的性質であり得る。 局所的症状は、 一般にアレルゲンが体内に入った位 置の上皮表面に起こる。 全身的効果は、 アナフィ ラキシーショ ックが含 まれ、 血管内での抗原に対する I g E—好塩基球応答の結果である。 ダニ、 各種花粉等の環境中の各種物質、 食物等に含まれる抗原性物質 がアレルゲンとして作用しているアレルギー性疾患が多い。 このなかで. 真菌を原因とするアレルギー性疾患も非常に多く、 カ ンジダ、 ァスペル ギルス、 アルタナリア、 クラ ドスポリゥム、 マラセチア、 ぺニシリウム 等に由来するアレルゲンが原因として働いている。 The combination of IgE and specific allergens causes the IgE to bridge on the cell surface, producing the physiological effects of the IgE-antigen interaction. These physiological effects involve what are called chemical mediators. Chemical mediators include those already contained in mast cells and eosinophil granules, which are released out of cells by degranulation upon activation, such as histamine, serotonin, and Some of them are newly synthesized by activation of mast cells and others, such as eosinophil factors. In the latter case, following the activation of Hosuhori lipase A 2, lipoxygenase, cyclo O key Shige kinase is activated, it acts on Araki Don acid membrane phospholipids, various leuco preparative Lien acids, thromboxanes etc. generated Is done. These chemical mediators cause allergic inflammation such as long-term contraction of bronchial smooth muscle and mucosal edema. These effects can be systemic or local in nature, depending on the route by which the antigen enters the body and the pattern of IgE deposition on mast cells or basophils. Local symptoms generally occur on the epithelial surface where the allergen has entered the body. Systemic effects, including anaphylactic shock, are the result of an IgE-basophil response to antigens in blood vessels. There are many allergic diseases in which various substances in the environment, such as mites and various pollens, and antigenic substances contained in foods, etc., act as allergens. Among them, there are many allergic diseases caused by fungi, and allergens derived from Candida, Aspergillus, Alternaria, Cladosporium, Malassezia, Penicillium, etc. work as a cause.
このような真菌による感染症、 汚染に対する治療、 防御の目的に使用 可能である抗真菌剤は、 現在のところ、 非常に少数のものが知られてい るに過ぎない- このうち、 特にヒ トを始めとする動物の全身性感染症に 対する治療剤としては、 アンホテリ シン B、 フルシ 卜シン、 ミ コナゾ一 ル、 フルコナゾ一ル等を挙げることができる。 し力、し、 これらのものは、 効力、 毒性、 抗菌スぺク トル等の点で問題があり、 治療剤と しては充分 なものではなかつた。 Only a very small number of antifungal agents are currently known that can be used to treat or defend against such fungal infections and contamination-especially humans. Other therapeutic agents for systemic infections in animals include amphotericin B, flucitrosin, and miconazo. And fluconazole. These compounds have problems in efficacy, toxicity, antibacterial spectrum, etc., and have not been sufficient as therapeutic agents.
また、 アレルギー性疾患の治療薬としては、 多種多様なものが存在し、 上述した各種ケミ カルメディエーターの産生を抑制するように働く リポ キシゲナーゼ阻害剤、 卜口ンボキサン合成酵素阻害薬薬剤 ； ケミカルメ ディエーターの効果を阻害するように働く抗ヒス夕 ミ ン薬、 ロイコ ト リ ェン ΰセプター阻害薬がある。 また、 ステロイ ド薬は、 ケミカルメディ エーターの産生を抑制するように働くほか、 各種生理活性を有しており、 抗ァレルギ一剤として最も重要なものである。 発明の要約  In addition, there are a wide variety of remedies for allergic diseases, and lipoxygenase inhibitors, which act to suppress the production of the various chemical mediators described above, drugs that act as toxin boxane synthase inhibitors, and chemical mediators. There are anti-histamine inhibitors and leukotriene perceptor inhibitors that work to inhibit their effects. Steroid drugs also act to suppress the production of chemical mediators and have various physiological activities, making them the most important antiallergic agents. Summary of the Invention
本発明の目的は、 上述の現状に鑑み、 真菌感染症、 アレルギー性疾患 及び免疫の治療剤として有用な新規生理活性物質を提供するところにあ 本発明者らは、 新規な生理活性物質の探索を目的として、 多数の微生 物を自然界より分離し、 その産生する生理活性物質を単離し、 生物学的 性質を調べたところ、 ぺニシリウム (P e n i c i I l i um) 属に厲 する微生物の培養物中に、 カンジダ、 ァスペルギルス、 ク リプ コッカ ス、 マラセチア等の病原性真菌に対して抗菌活性を示す生理活性物質が 存在していることを見いだした。 その後、 本発明者らは、 これらの生理 活性物質を単離し、 その理化学的性質を調べた結果、 特有の理化学的性 質を有する文献未記載の新規物質であることを確認し、 これらの生理活 性物質を TKR 1 7 8 5 (以下、 「TKR 1 7 8 5— 1」 とする） 及び T KR 1 7 8 5 - I I (以下、 これらをまとめて （"TKR 1 7 8 5類」 という） と命名した。 更に、 TKR 1 7 8 5類-がアレルギー反応に関与 する酵素の阻害活性を始めとして、 免疫系への生理活性を有することを 見いだし、 本発明を完成するに至った。 An object of the present invention is to provide a novel bioactive substance useful as a therapeutic agent for fungal infectious diseases, allergic diseases, and immunity in view of the above-mentioned situation. For the purpose of this study, we isolated many microorganisms from nature, isolated the biologically active substances produced by them, and examined their biological properties.The culture of microorganisms belonging to the genus Penici Ilium It was found that a biologically active substance showing antibacterial activity against pathogenic fungi such as Candida, Aspergillus, Clipcoccus and Malassezia was present in the product. Subsequently, the present inventors isolated these physiologically active substances and examined their physicochemical properties. As a result, they confirmed that they were novel substances having unique physicochemical properties and not described in the literature. The active materials are TKR 1785 (hereinafter referred to as “TKR 1785—1”) and TKR 1785-II (hereinafter collectively referred to as “TKR 1785 class”). In addition, TKR 1785-classes are involved in allergic reactions The present inventors have found that the enzyme has a physiological activity on the immune system, including the inhibitory activity of the enzyme, and have completed the present invention.
すなわち、 第一の本発明は、 下記一般式 （A) ；  That is, the first present invention provides the following general formula (A):
OH OH R CH2OH OH OH R CH 2 OH
I I I I £ IIII £
H3C-(GH2)i2-CH-CH2-CH-CH— CH2-CO-NH-CH-CO-NH-CH-CH2OHH 3 C- (GH 2 ) i2-CH-CH 2 -CH-CH— CH 2 -CO-NH-CH-CO-NH-CH-CH 2 OH
H2 H 2
(A)  (A)
(式中、 Rは、 一 CH (CH3 ) 2 又は— CH (CHa ) C2 Hs であ る。 ） で表される新規な生理活性物質 TKR 1 7 8 5類である。 (Wherein, R is one CH (CH 3 ) 2 or —CH (CHa) C 2 H s ).
第二の本発明は、 ぺニシリウム (P e n i c i I l i um) 厲に属す る菌株であって、 生理活性物質 TKR 1 7 8 5類を産生する菌株を培養 し、 その後、 前記菌株の培養物から目的物を単離する生理活性物質 TK R 1 7 8 5類の製造方法である。  A second aspect of the present invention is to culture a strain belonging to Penicillium (Penici Ilium), which produces a physiologically active substance TKR1785, and thereafter, from the culture of the strain, This is a method for producing a physiologically active substance TKR1785 for isolating a target substance.
第三の本発明は、 ベニンリウム (P e n i c i 1 1 i u m) 属に厲し、 生理活性物質 TKR 1 7 8 5類を産生する微生物である。  The third invention is a microorganism which belongs to the genus Beninium (Penic11ium) and produces the physiologically active substances TKR1785.
第四の本発明は、 生理活性物質 TKR 1 7 8 5類を含有する医薬組成 物である。 図面の簡単な説明  The fourth invention is a pharmaceutical composition containing the physiologically active substance TKR1785. BRIEF DESCRIPTION OF THE FIGURES
図 1は、 生理活性物質 TKR 1 7 8 5 - 1の紫外線吸収スぺク トルを 示す図である。 縦軸は波長 （nm) を示す。  FIG. 1 is a diagram showing an ultraviolet absorption spectrum of the physiologically active substance TKR1785-1. The vertical axis indicates the wavelength (nm).
図 2は、 生理活性物質 TKR 1 7 8 5 - 1の赤外線吸収スぺク トルを 示す図である。 横軸は波数 （ c m_') を示す。  FIG. 2 is a diagram showing an infrared absorption spectrum of the physiologically active substance TKR1785-1. The horizontal axis shows the wave number (cm_ ').
図 3は、 生理活性物貿 T K R 1 7 8 5— Iの 'Η— NMRスペク トル を示す図である。 横軸は化学シフ ト値 （p p m) を示す。 図 4は、 生理活性物質 TKR 1 7 8 5— 1の'3じー1^1^1¾スぺク トル を示す図である。 横軸は化学シフ ト値 （P pm) を示す。 FIG. 3 is a diagram showing a 'Η-NMR spectrum of TKR1785-I, a biologically active substance. The horizontal axis shows the chemical shift value (ppm). Figure 4 is a diagram showing a physiologically active substance TKR 1 7 8 5- 1 '3 G 1 ^ 1 ^ 1¾ spectrum. The horizontal axis shows the chemical shift value (P pm).
図 5は、 生理活性物質 TKR 1 7 8 5 — Iの H P L Cでの溶出位置を 示す図である。 縦軸は保持時間 （分） を示し、 横軸は相対紫外吸収強度 を示す。  FIG. 5 is a diagram showing the elution position of the physiologically active substance TKR1785-I in HPLC. The vertical axis indicates the retention time (minutes), and the horizontal axis indicates the relative ultraviolet absorption intensity.
図 6は、 生理活性物質 TKR 1 7 8 5— I Iの 'H— NMRスべク ト ルを示す図である。 横軸は化学シフ ト値 （P Pm) を示す。  FIG. 6 is a diagram showing an 'H-NMR spectrum of the physiologically active substance TKR1785-II. The horizontal axis shows the chemical shift value (P Pm).
図 7は、 生理活性物質 TKR 1 7 8 5 — 1 1の13〇ー 1^1¾スぺク ト ルを示す図である。 横軸は化学シフ ト値 （P pm) を示す。 Figure 7 is a biologically active substance TKR 1 7 8 5 - a diagram showing the 1 1 of 13 〇 over 1 ^ 1¾-spectrum Le. The horizontal axis shows the chemical shift value (P pm).
図 8は、 生理活性物質 TKR 1 7 8 5 — I Iの H P L Cでの溶出位置 を示す図である。 縦軸は保持時間 （分） を示し、 横軸は相対紫外吸収強 度を示す。 発明の詳細な開示  FIG. 8 is a diagram showing the elution position of the physiologically active substance TKR1785-II in HPLC. The vertical axis indicates the retention time (minutes), and the horizontal axis indicates the relative ultraviolet absorption intensity. Detailed Disclosure of the Invention
生理活性物質 TKR 1 7 8 5— Iは、 下記 （ 1 ) 、 （ 2 ) 、 （ 3 ) 、 (4 ) 、 （ 5 ) 、 （6 ) 及び （ 7 ) の理化学的性質を有する。  The physiologically active substance TKR1785-I has the following physicochemical properties (1), (2), (3), (4), (5), (6) and (7).
( 1 ) F A B— MS法による質量スぺク トルが、 mZ z 5 1 8 [M + H] のピークを有する。  (1) Mass spectrum by FAB-MS method has a peak of mZz518 [M + H].
( 2 ) 分子式が、 C27H55N3 0である。 (2) The molecular formula is C 27 H 55 N 30 .
( 3 ) メタノール中における紫外線吸収スぺク トルが、 図 1に示すとお り末端吸収を示す。  (3) The ultraviolet absorption spectrum in methanol shows terminal absorption as shown in FIG.
( 4 ) K B r法による赤外線吸収スぺク トルの主要な吸収波数が、 3 4 1 0 c m―'、 2 9 2 0 c m— '、 2 8 5 0 cm— '、 1 6 7 0 c m一'、 1 5 4 0 c m— '、 1 4 7 0 c m— '、 1 2 1 0 c m"', 1 1 4 0 c m '、 1 0 5 0 c m I、 8 4 0 c m— '、 8 0 0 c m 7 2 0 c m-1である。 (4) The main absorption wave numbers of the infrared absorption spectrum by the KBr method are 3410 cm- ', 2920 cm-', 2850 cm- ', 1670 cm- ', 1540 cm—', 1470 cm— ', 1 210 cm "', 1140 cm ', 1500 cm I, 8400 cm—', 800 cm 720 cm- 1 .
( 5 ) メタノール、 水に可溶であり、 クロ口ホルム、 へキサンに難溶で め O o (5) Soluble in methanol and water, poorly soluble in chloroform and hexane O O
(6 ) 図 3に示す 'H— NMRスぺク トル、 図 4に示す'3 C—： \' M Rス ぺク トルを有する。 (6) shown in FIG. 3 'H- NMR spectrum, shown in Figure 4' 3 C-: having \ 'MR scan Bae-vector.
( 7 ) 逆相分配高速液体ク口マ トグラフィ一において図 5に示す位置に 溶出される特性を有する。  (7) It has the property of being eluted at the position shown in Fig. 5 in reversed-phase partition high-performance liquid chromatography.
これらの性質を基に構造解析を行い、 TKR 1 7 8 5— Iは、 式 （ I ) に示す化学構造を有していることが明らかになった。  Structural analysis was performed based on these properties, and it was revealed that TKR1785-I had the chemical structure shown in formula (I).
OH OH CH(CH3)2 CH2OH OH OH CH (CH 3 ) 2 CH 2 OH
H3C-(CH2)i2-CH-CH2-CH-CH— CH2-CO-NH-CH-CO-NH-CH-CH2OH H 3 C- (CH 2) i 2 -CH-CH2-CH-CH- CH 2 -CO-NH-CH-CO-NH-CH-CH 2 OH
NH2 (L) NH 2 (L)
(I)  (I)
生理活性物質 TKR 1 7 8 5— I Iは、 下記 （ 8 ) 、 （ 9 ) 、 (10) 及 び （11) の理化学的性質を有する。 The physiologically active substance TKR1785-II has the following physicochemical properties (8), (9), (10) and (11).
( 8 ) F A B—M S法による質量スぺク トルが、 m_ z 5 3 2 :M H] — のピークを有する。  (8) The mass spectrum according to the FAB-MS method has a peak of m_z532: MH] —.
(9) 分子式が、 C2BH「,7N3 0である。 (9) The molecular formula is C 2 B H “, 7 N 30 .
(10) 図 6に示す H— NMRスぺク トル、 図 7に示す'3 C— NMRス ぺク トルを有する。 (10) It has the H-NMR spectrum shown in FIG. 6 and the ' 3 C-NMR spectrum shown in FIG.
(11) 逆相分配高速液体ク口マ トグラフィ一において図 8に示す位置に 溶出される特性を有する。  (11) It has the property of being eluted at the position shown in Fig. 8 in reversed phase distribution high performance liquid chromatography.
これらの性質を基に構造解析を行い、 生理活性物質 TKR 1 7 8 5 - I Iは、 式 （ I I ) に示す化学構造を有していることが明らかになつた。 CH3 Structural analysis was performed based on these properties, and it was revealed that the physiologically active substance TKR1785-II had the chemical structure represented by the formula (II). CH 3
I I
OH OH CH-C2H5 CH2OH OH OH CH-C2H5 CH 2 OH
H3C-(CH2)12-CH- CH2- CH— CH— CH2-CO -NH_ CH- CO -ΝΉ- CH_CH2OH H 3 C- (CH 2 ) 12 -CH- CH 2 -CH— CH— CH 2 -CO -NH_ CH- CO -ΝΉ- CH_CH 2 OH
t (L) t (L)
H2 H 2
(H)  (H)
上記 TKR 1 7 8 5類は、 ぺニシリウム (Penicillium ) 属に厲し、 上記 TKR 1 7 8 5を産生する菌株を培養し、 その後、 上記菌株の培養 物から単離することにより製造することができる。 The TKR1785 can be produced by culturing a strain that produces the TKR1785 in the genus Penicillium, and then isolating the strain from a culture of the strain. it can.
本発明で使用される上記菌株としては、 ぺニシリウム (Penicillium ) 厲に属し、 上記 TKR 1 7 8 5類を産生するものであれば特に限定され ず、 例えば、 ぺニシリウム · エスピー （Penicillium sp. ) TKR 1 7 8 5株 （以下、 「TKR 1 7 8 5株」 という） 等を挙げることができる。 上記 TKR 1 7 8 5株は、 文献未記載の新菌株であって、 本発明者ら によって、 兵庫県にて採取された試料中より初めて分離同定されたもの であり、 TKR 1 7 8 5類を有利に産生する特性を有するものである。 以下、 上記 TKR 1 7 8 5株の菌学的性質を詳細に説明する。  The strain used in the present invention is not particularly limited as long as it belongs to Penicillium and produces the above-mentioned TKR1785s. For example, Penicillium sp. TKR 175 strains (hereinafter referred to as "TKR 175 strains"). The above-mentioned TKR1785 strain is a new strain not described in the literature, and was isolated and identified by the present inventors for the first time from a sample collected in Hyogo Prefecture. Has the property of advantageously producing Hereinafter, the mycological properties of the TKR1785 strain will be described in detail.
上記 TKR 1 7 8 5株の各種培地におけるコロニー （以下、 J集落 j ともいう） の色調は、 表 1 に示すとおりである。 なお、 表中の色調は、 日本工業規格 J I S Z 8 1 0 2 ( 1 9 8 5年） による色名を基準と し、 培地に接種後、 2 5でで培養し、 7 曰後に観察した結果によって示 したものである。 表 1 Table 1 shows the color tone of the colonies (hereinafter, also referred to as J colonies j) of the above TKR1785 strains in various media. In addition, the color tone in the table is based on the color name according to Japanese Industrial Standard JISZ 8102 (1989), after inoculating the medium, culturing at 25, and observing after 7 It is shown. table 1
Figure imgf000010_0001
Figure imgf000010_0001
上記 TKR 1 7 8 5株は、 麦芽エキス寒天培地、 ポテ トデキス トロー ス寒天培地、 ッァペック寒天培地等で速やかに生育し、 そのコロニーの 表面はビロー ド状で、 中心部はやや隆起している。 TKR 1 7 8 5株の 分生子柄は、 9 0〜 2 7 0 x し 8〜 3. 0 mの滑面で、 ほとんど複 輪生体対称型のベニシリを形成する。 メ ト レは 1 2. 0〜 1 4. 0 2. 8〜 3. 2 〃 で 2〜 4本が群生し、 フ ィ アライ ドは 9. 0 ~ 1 0. 0 1 . 8〜 2. 4 imで輪生状となる。 分生子は滑面の球形か ら亜球形で、 そのサイズは 2. 2〜 3. 2 x 2. 4〜 4. 0 mである c 上記 TKR 1 7 8 5株の菌学的性質のうち、 生理学的性質は、 下記に 示す通りである。 The TKR1785 strain grows rapidly on a malt extract agar medium, potato dextroth agar medium, Wapec agar medium, and the like, and the surface of the colony is in the form of a belode, and the center is slightly raised. The conidiophores of the TKR 1785 strain have a smooth surface of 90 to 270 x 8 to 3.0 m, and form almost bicyclic biosymmetric Benicili. The metres are 12.0 to 14.0 2.8 to 3.2 2, and 2 to 4 trees are clustered, and the fireride is 9.0 to 10.0 1.8 to 2.4. It becomes a ring shape in im. Conidia globose or et subglobose the smooth surface, the size of 2. 2~ 3. 2 x 2. 4~ 4. Of mycological properties of c the TKR 1 7 8 5 strains is 0 m, Physiological properties are as shown below.
生育温度範囲 ： 生育可能温度範囲が、 1 0〜 3 0てであり、 生育最適温 度が、 2 5 °C付近である。 Growth temperature range: The temperature range where growth is possible is 10-30, and the optimum growth temperature The temperature is around 25 ° C.
生育 P H範囲 ： 生育可能 p H範囲が、 p H 3〜p H 9であり、 生育最適 p H力 、 p H 5付近である。 Growth pH range: The range of pH that can be grown is pH 3 to pH 9, and the growth optimal pH is around pH 5.
上述の菌学的性質を有する菌種を、 カルロス ' ラ ミ レツ （ C a r 1 0 s A bacterial species having the above-mentioned mycological properties is referred to as Carlos' Ramirez (Car 10 s).
R am i r e z ) 著、 マニュアル ' アン ド ' ア トラス ' ォブ ' ザ ' ベニ シ リ ア （ M a n u a l a n d a t l a s o f t h e P e n i c i 1 1 i a ) 、 エルセピア ' バイ オメ ディ カル ' プレス (E l s e v i e r B i o m e d i c a l P r e s s ) ( 1 9 8 2 年） 等の文献に記載されたぺニシリゥム属の菌種について検索すること により、 上記 TKR 1 7 8 5株は、 ベニシリゥム厲に属する菌株である と同定することができる。 R am irez), Manuals' And 'Atlas' Job' The 'Benicilia (ManualandatlasofthePenici 11 ia), El Sepia' Biomedical 'Press (Elsevier Biomedical Press) The above TKR1785 strain can be identified as a strain belonging to Benicillium by searching for strains of the genus Penicillium described in the literature such as 1982.
しかしながら、 ぺニシリゥ厶厲に厲する菌株であって、 TKR 1 7 8 5類の産生能を有するものについては、 これまで報告がなされたことは ない。 そこで、 本発明 ¾らは、 これを新菌株とし、 ぺニシリウ厶 ' エス ピ— TKR 1 7 8 5株 （P e n i c i 1 1 i urn s p . TKR 1 7 8 5 ) と命名し、 P e n i c i 1 1 i u m s p. TKR 1 7 8 5と表示 し、 通商産業省工業技術院生命工学工業技術研究所 〔あて名 ； 日本国茨 城県つく ば市東 1丁目 1番 3号 （郵便番号 3 0 5 ) 〕 に、 寄託番号 F E RM B P— 5 7 8 8 (原寄託日 ； 平成 7年 5月 1 7 日、 国際寄託 への移管請求日 ；平成 9年 1月 1 7日） として寄託した。  However, there has been no report on a strain of D. niger, which has the ability to produce TKR1785. Therefore, the present inventors designated this strain as a new strain, and named it as Penicillium sp. Strain TKR1785 (Penici 11 i urn sp. TKR1785). iums p. TKR 1 785 5 and the Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology, Institute of Biotechnology and Industrial Technology [Address: 1-3-1, Higashi, Tsukuba, Ibaraki, Japan (zip code: 3005)] Deposit No. FE RM BP—5788 (Original deposit date; May 17, 1995, request date for transfer to international deposit; January 17, 1997).
本発明においては、 上記 T K R 1 7 8 5株の他に、 TKR 1 7 8 5株 の自然的又は人工的変異株、 その他のぺニシリゥム属に属する菌種等で あって、 TKR 1 7 8 5類の産生能を有する微生物を使用することがで きる。  In the present invention, in addition to the above-mentioned TKR 1785 strain, natural or artificial mutant strains of the TKR 1785 strain, other strains belonging to the genus Penicillium, etc. Microorganisms having the ability to produce such species can be used.
上記微生物の上記 TKR 1 7 8 5類の生産性は、 ぺニシリ ゥム厲の菌 株の適当な培養物中に含まれる生理活性物質について、 図 5 と同様の条 件で逆相分配高速液体ク口マ トグラフィ一に供し、 溶出位置を測定する こと、 フ ォ 卜ダイオー ドァレイ装置等を用いた紫外線吸収スぺク トルの 測定を行うこと、 上記 T K R 1 7 8 5類に関する理化学的性質と比較す ること、 更に必要に応じて、 上記逆相分配高速液体ク口マ トグラフ ィ ー により溶出した溶出物について適当な分子量測定装置で分子量測定を行 うことにより、 容易に決定することができる。 The productivity of the above-mentioned TKR1785 class of the above-mentioned microorganisms is the same as that of Fig. 5 for physiologically active substances contained in an appropriate culture of a strain of Nissan strain. Subject to reversed phase distribution high-performance liquid chromatography in order to determine the elution position, and to measure the ultraviolet absorption spectrum using a photo diode array device, etc. By comparing with the physicochemical properties of chemicals and, if necessary, by conducting a molecular weight measurement on the eluted material by the reversed-phase partition high-performance liquid chromatography described above using an appropriate molecular weight measurement device. Can be determined.
本発明においては、 丁 K R 1 7 8 5類は、 上記 T K R 1 7 8 5類を産 生する菌株を、 栄養源含有培地に接種し、 培養することによって製造さ れる。 上記栄養源のうち、 炭素源としては、 例えば、 グルコース、 フル ク ト一ス、 サッカロース、 澱粉、 デキス ト リ ン、 グリセリ ン、 糖蜜、 水 飴、 油脂類、 有槻酸等を挙げることができる。  In the present invention, the KR1785s are produced by inoculating a strain producing the TKR185s into a nutrient-containing medium and culturing the same. Among the above nutrient sources, examples of the carbon source include glucose, fructos, saccharose, starch, dextrin, glycerin, molasses, syrup, oils and fats, Aritsuki acid and the like. .
上記栄養源のうち、 窒素源としては、 例えば、 大豆粉、 綿実粉、 コー ンスチ一ブリ カー、 カゼイ ン、 ペプト ン、 酵母エキス、 肉エキス、 胚芽、 尿素、 アミ ノ酸、 アンモニゥム塩等の有機窒素化合物、 無機窒素化合物 等を挙げることができる。 上記栄養源のうち、 塩類としては、 例えば、 ナ ト リウム塩、 カリウム塩、 カルシウム塩、 マグネシウム塩、 りん酸塩 等の無機塩類を挙げることができる。 これらはそれぞれ単独で使用され てもよく、 適宜組み合わせて使用されてもよい。  Among the above nutrient sources, nitrogen sources include, for example, soybean flour, cottonseed flour, corn brik, casein, peptone, yeast extract, meat extract, germ, urea, amino acid, ammonium salt, etc. Organic nitrogen compounds, inorganic nitrogen compounds and the like can be mentioned. Among the above nutrient sources, examples of the salt include inorganic salts such as sodium salt, potassium salt, calcium salt, magnesium salt, and phosphate. Each of these may be used alone or in combination as appropriate.
上記栄養源含有培地には、 更に必要に応じて、 鉄塩、 銅塩、 亜鉛塩、 コバル卜塩等の重金属 ； ビォチン、 ビタ ミ ン B , 等のビタ ミ ン類 ； その 他、 菌の生育を助け、 T K R 1 7 8 5類の産生を促進する有機物、 無機 物等を適宜添加することができる。  The nutrient-containing medium may further contain, if necessary, heavy metals such as iron salts, copper salts, zinc salts and cobalt salts; vitamins such as biotin and vitamin B; Organic substances, inorganic substances, and the like that promote the production of TKR1785 can be added as appropriate.
上記栄養源含有培地には、 上記栄養源の他に、 更に必要に応じて、 シ リ コ一ンオイル、 ポリアルキレングリ コールエーテル等の消泡剤、 界面 活性剤等を添加することができる。  In addition to the nutrient source, an antifoaming agent such as silicone oil and polyalkylene glycol ether, a surfactant, and the like can be added to the nutrient source-containing medium, if necessary.
上記 T K R 1 7 8 5類を産生する菌株を、 上記栄養源含有培地で培養 するに際しては、 生理活性物質の産生を微生物の培養によって行う際に 一般的に使用される方法を採用することができるが、 液体培養法、 なか でも振逢又は深部通気損拌培養法を好適に使用することができる。 Culture the above strains producing TKR1785 in the nutrient-containing medium In this case, a method generally used for producing a physiologically active substance by culturing a microorganism can be employed, but a liquid culture method, in particular, a shaking or deep aeration stirring culture method is preferable. Can be used.
上記培養は、 1 5〜 2 5てで行うことが好ましく、 培地の p Hは、 通 常 p H 3〜 8であるが、 p H 5付近であることが好ましい。 培養期間は、 通常 3〜 1 1 日で充分な産生量を得ることができる。  The above culture is preferably performed at 15 to 25, and the pH of the medium is usually pH 3 to 8, but is preferably around pH 5. In general, a sufficient production amount can be obtained in a culture period of 3 to 11 days.
上述の培養方法によって、 T K R 1 7 8 5類は、 培養液及び菌体に含 有されて培養物中に蓄積される。 本発明においては、 培養物中に蓄積さ れた T K R 1 7 8 5類は、 これら生理活性物質の理化学的性質及び生物 学的性質を利用して培養物中から分離した後、 必要に応じて更に精製し、 取得することができる。  By the above-described culture method, TKR1785 is contained in the culture solution and the cells and accumulates in the culture. In the present invention, the TKR1785 accumulated in the culture is separated from the culture using the physicochemical and biological properties of these physiologically active substances, and then, if necessary, It can be further purified and obtained.
上記分離は、 培養物全体を、 酢酸ェチル、 酢酸プチル、 クロ口ホルム、 ブ夕ノール、 メチルイソブチルケトン等の非親水性有機溶媒で抽出する ことにより行うことができる。 また、 培養物を濾過又は遠心分離によつ て培養液と菌体とに分離した後、 培養液、 菌体のそれぞれから分離する こともできる。  The above separation can be carried out by extracting the whole culture with a non-hydrophilic organic solvent such as ethyl acetate, butyl acetate, black form, butanol, and methyl isobutyl ketone. Further, after the culture is separated into a culture solution and cells by filtration or centrifugation, the culture can be separated from each of the culture solution and the cells.
上記培養液から T K R 1 7 8 5類を分離するには、 上記非親水性有機 溶媒で抽出する方法を採用することもでき、 また、 培養液を吸着性の担 体に接触させ、 培養液中の T K R 1 7 8 5類を担体に吸着させた後、 溶 媒で溶出する方法を採用することもできる。 上記担体としては、 例えば- 活性炭、 粉末セルロース、 吸着性榭脂等を挙げることができる。 上記溶 媒としては、 担体の種類、 性質等によって適宜 1種又は 2種以上を組み 合わせて使用することができ、 例えば、 含水アセ ト ン、 含水アルコール 類等の水溶性有機溶媒の含水溶液等を適宜組み合わせたもの等を挙げる ことができる。 上記菌体から T K R 1 7 8 5類を分離するには、 ァセ ト ン等の親水性有機溶媒で抽出する方法を採用することができる- 本発明においては、 このようにして培養物中から分離された T K R ' 1 7 8 5類の粗抽出物を、 必要に応じて、 更に精製する工程に付するこ とができる。 上記精製は、 脂溶性生理活性物質の分離、 精製に通常使用 される方法によって行うことができ、 このような方法としては、 例えば、 シリカゲル、 活性アルミナ、 活性炭、 吸着性樹脂等の担体を用いるカラ ムクロマトグラフィー法、 高速液体クロマ トグラフィ一法等を挙げるこ とができる。 シリ力ゲルを担体として用いるカラムクロマ トグラフィ ー 法を採用する場合は、 溶出溶媒としては、 例えば、 クロ口ホルム、 酢酸 ェチル、 メ タノール、 アセ トン、 水等を挙げることができ、 これらは 2 種以上を併用することができる。 In order to separate TKR1785 from the culture solution, a method of extracting with the above-mentioned non-hydrophilic organic solvent can be adopted.Also, the culture solution is brought into contact with an adsorptive carrier, and Alternatively, a method may be employed in which TKR1785 is adsorbed on a carrier and then eluted with a solvent. Examples of the carrier include activated carbon, powdered cellulose, and adsorbent resin. The solvent may be used alone or in combination of two or more depending on the type and properties of the carrier.For example, an aqueous solution of a water-soluble organic solvent such as hydrated acetate or hydrated alcohol may be used. And the like in combination. In order to separate TKR1785 from the above cells, a method of extraction with a hydrophilic organic solvent such as acetone can be adopted. In the present invention, the crude extract of TKR'1785s thus separated from the culture can be subjected to a step of further purification, if necessary. The above-mentioned purification can be carried out by a method usually used for separation and purification of a fat-soluble physiologically active substance. Examples of such a method include a method using a carrier such as silica gel, activated alumina, activated carbon, and an adsorbent resin. And high-performance liquid chromatography. When column chromatography using silylation gel as a carrier is used, examples of the eluting solvent include chromatographic form, ethyl acetate, methanol, acetone, water, and the like. Can be used in combination.
上記高速液体ク口マ トグラフィ一法を採用する場合は、 担体としては、 例えば、 ォクタデシル基、 ォクチル基、 フ Xニル基等が結合した化学結 合型シリカゲル； ポリスチレン系ポーラスボリマーゲル等を挙げること ができ、 移動相としては、 例えば、 含水メ タノール、 含水ァセ 卜ニ ト リ ル等の水溶性有機溶媒の含水溶液等を使用することができる。  When the above-described high performance liquid chromatography is employed, examples of the carrier include chemically bonded silica gel having an octadecyl group, an octyl group, and a phenyl group bonded thereto; a polystyrene-based porous polymer gel; As the mobile phase, for example, an aqueous solution of a water-soluble organic solvent such as hydrated methanol and hydrated acetonitrile can be used.
本発明の T K R 1 7 8 5類は、 そのまま、 又は、 その薬理学的に許容 される塩として医薬に使用することができる。 上記 T K R 1 7 8 5類、 又は、 その薬理学的に許容される塩を含有する医薬組成物としては特に 限定されず、 例えば、 抗真菌剤、 抗アレルギー剤、 免疫調節剤等を挙げ ることができる。 本発明においては、 抗アレルギー剤、 免疫調節剤等と して使用されることが好ま しいが、 用途はこれらに限定されるものでは なく、 その他の用途に用いる場合であっても、 上記 T K R 1 7 8 5類、 又は、 その莱理学的に許容される塩を含有する医薬組成物はすべて、 本 発明に含まれる。  The TKR1785s of the present invention can be used in medicine as it is or as a pharmacologically acceptable salt thereof. The pharmaceutical composition containing the above-mentioned TKR 17885 or a pharmacologically acceptable salt thereof is not particularly limited, and examples thereof include antifungal agents, antiallergic agents, and immunomodulators. Can be. In the present invention, it is preferable to use as an antiallergic agent, an immunomodulator, etc., but the use is not limited to these. The present invention includes all pharmaceutical compositions containing 875 or a reasonably acceptable salt thereof.
上記塩としては薬理学的に許容されるものであれば特に限定されず、 例えば、 塩酸、 硫酸、 硝酸、 りん酸、 ふつ化水素酸、 臭化水素酸等の鉱 酸の塩 ； ぎ酸、 酢酸、 酒石酸、 乳酸、 クェン酸、 フマール酸、 マレイ ン 酸、 こはく酸、 メ タ ンスルホン酸、 エタ ンスルホン酸、 ベンゼンスルホ ン酸、 トルエンスルホン酸、 ナフタ レンスルホン酸、 カ ンフ ァースルホ ン酸等の有機酸の塩 ； ナ ト リ ウム、 カ リ ウム、 カルシウム等のアルカ リ 金属又はアル力 リ土類金属等の塩等を挙げることができる。 The salt is not particularly limited as long as it is pharmacologically acceptable, and examples thereof include minerals such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrofluoric acid, and hydrobromic acid. Salts of acids; formic acid, acetic acid, tartaric acid, lactic acid, citric acid, fumaric acid, maleic acid, succinic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalenesulfonic acid, carboxylic acid Salts of organic acids such as dimethyl sulfonate; salts of alkali metals such as sodium, potassium and calcium, and salts of alkaline earth metals.
本発明の T K R 1 7 8 5類、 又は、 その薬理学的に許容される塩を医 薬として投与する場合、 本発明の T K R 1 7 8 5類、 又は、 その薬理学 的に許容される塩は、 そのまま、 又は、 医薬的に許容される無毒かつ不 活性の担体中に、 例えば、 0 . 1〜 9 9 . 5 %、 好ま しく は 0 . 5〜 9 0 %含有する医薬組成物、 例えば、 抗真菌剤、 抗アレルギー剤、 免疫 調節剤等として、 ヒ トを含む動物に投与される。  When administering the TKR1785 class of the present invention or a pharmacologically acceptable salt thereof as a medicament, the TKR1785 class of the present invention or a pharmacologically acceptable salt thereof is used. Is a pharmaceutical composition containing 0.1 to 99.5%, preferably 0.5 to 90%, as it is, or in a pharmaceutically acceptable non-toxic and inert carrier. It is administered to human-containing animals as an antifungal agent, an antiallergic agent, an immunomodulator and the like.
上記担体としては、 例えば、 固形、 半固形若しくは液状の希釈剤、 充 塡剤又はその他の処方用の助剤等を挙げることができ、 これらは、 1種 以上を用いることができる。  Examples of the carrier include solid, semi-solid or liquid diluents, fillers, and other auxiliaries for formulation. One or more of these can be used.
上記医薬組成物は、 投与単位形態で投与することが好ま しく、 経口投 与、 組織内投与、 局所投与 （経皮投与等） 又は経直腸的に投与すること ができる。 上記医薬組成物は、 これらの投与方法に適した剤型で投与さ れることは当然である。  The above pharmaceutical composition is preferably administered in the form of a dosage unit, and can be administered orally, intraosseously, topically (such as transdermally) or rectally. Naturally, the above pharmaceutical composition is administered in a dosage form suitable for these administration methods.
本発明の T K R 1 7 8 5類、 又は、 その薬理学的に許容される塩を医 薬として投与する場合、 抗真菌剤、 抗アレルギー剤又は免疫調節剤とし ての用量は、 年齢、 体重等の患者の状態、 投与経路、 病気の性質と程度 等を考慮した上で調整することが望ましいが、 通常は、 ヒ 卜については. 成人に対して本発明の T K R 1 7 8 5類、 又は、 その薬理学的に許容さ れる塩の有効成分量として、 一日当たり、 1 0〜 2 0 0 0 m gの範囲で ある。 上記範囲未満の用量で足りる場合もあるが、 逆に上記範囲を超え る用量を必要とする場合もある。 多量に投与するときは、 一日数回に分 割して投与することが望ま しい。 When the TKR1785 or the pharmacologically acceptable salt thereof of the present invention is administered as a medicine, the dose as an antifungal agent, an antiallergic agent or an immunomodulator is determined by the age, body weight, etc. It is desirable to make adjustments in consideration of the condition of the patient, the administration route, the nature and extent of the disease, etc., but usually, for humans. For adults, the TKR1775-class of the present invention, or The amount of the active ingredient of the pharmacologically acceptable salt is in the range of 10 to 2000 mg per day. Dosages below the above range may be sufficient, while conversely, doses above the above range may be required. When administering large amounts, take several times a day. It is advisable to split the dose.
上記経口投与は、 固形、 扮末又は液状の用量単位で行うことができ、 例えば、 末剤、 散剤、 錠剤、 糖衣剤、 カブセル剤、 ドロップ剤、 舌下剤、 その他の剤型等により行うことができる。 例えば、 末剤は、 本発明の T K R 1 7 8 5類、 又は、 その薬理学的に許容される塩を適当な細かさに することにより製造される。 散剤は、 本発明の T K R 1 7 8 5類、 又は、 その薬理学的に許容される塩を適当な細かさとし、 ついで同様に細かく した医薬用担体、 例えば、 澱粉、 マンニ トール等の可食性炭水化物、 そ の他と混合することにより製造される。 必要に応じて、 嬌味剤、 保存剤、 分散剤、 着色剤、 香料等を混合してもよい。  The above oral administration can be carried out in solid, powder or liquid dosage units, for example, powders, powders, tablets, dragees, capsules, drops, sublinguals, other dosage forms, etc. it can. For example, a powder can be produced by appropriately preparing the TKR1785 class of the present invention or a pharmacologically acceptable salt thereof. The powder may be a finely-divided TKR1775-class of the present invention or a pharmaceutically acceptable salt thereof, and then a finely divided pharmaceutical carrier, for example, edible carbohydrates such as starch and mannitol. It is manufactured by mixing with others. If desired, flavoring agents, preservatives, dispersing agents, coloring agents, flavors and the like may be mixed.
上記非経口投与は、 例えば、 溶液や懸衝剤等の皮下、 筋肉内又は静脈 内注射用の液状用量単位形態を用いることによって行うことができる。 これらのものは、 本発明の T K R 1 7 8 5類、 又は、 その薬理学的に許 容される塩の一定量を、 例えば、 水性や油性の媒体等の注射の目的に適 合する非毒性の液状担体に懸濁又は溶解し、 次いで該懸澳液又は溶液を 滅菌することにより製造される。  Parenteral administration can be accomplished, for example, by using a liquid dosage unit form for subcutaneous, intramuscular or intravenous injection, such as a solution or a bumper. These compounds can be used to convert a certain amount of TKR1785 of the present invention or a pharmacologically acceptable salt thereof into, for example, an aqueous or oily vehicle suitable for injection purposes. It is manufactured by suspending or dissolving in a liquid carrier, and then sterilizing the suspension or solution.
上記局所投与 （経皮投与等） は、 例えば、 液、 ク リーム、 粉末、 ベー ス ト、 ゲル、 軟膏剤等の外用製剤の形態を用いることによって行うこと ができる。 これらのものは、 本発明の T K R 1 7 8 5類、 又は、 その薬 理学的に許容される塩の一定量を、 外用製剤の目的に適合する香料、 着 色料、 充填剤、 界面活性剤、 保湿剤、 皮膚軟化剤、 ゲル化剤、 担体、 保 存剤、 安定剤等のうちの一種以上と組み合わせることにより製造される c 直腸投与は、 本発明の T K R 1 7 8 5類、 又は、 その薬理学的に許容 される塩の一定量を、 例えば、 パルミチン酸ミ リスチルエステル等の高 級エステル類、 ボリエチレングリ コール、 カカオ脂、 これらの混合物等 の低融点の固体に混入した座剤等を用いて行うことができる。 発明を実施するための最良の形態 The above-mentioned topical administration (such as transdermal administration) can be carried out, for example, by using external preparations such as liquids, creams, powders, bases, gels, and ointments. These compounds can be used to convert a certain amount of TKR1785 of the present invention or a pharmaceutically acceptable salt thereof into fragrances, coloring agents, fillers, surfactants suitable for the purpose of external preparations , moisturizers, emollients, gelling agents, carriers, save agent, c rectal administration is prepared by combining one or more of such stabilizers, TKR 1 7 8 5 such present invention, or, An aliquot of the pharmacologically acceptable salt is mixed with low-melting solids such as higher esters such as myristyl palmitate, polyethylene glycol, cocoa butter, and mixtures thereof. It can be performed using an agent or the like. BEST MODE FOR CARRYING OUT THE INVENTION
以下に実施例を掲げて本発明を更に詳しく説明するが、 本発明はこれ らのみに限定されるものではない。 実施例 1  Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto. Example 1
TKR 1 7 8 5株 （F E RM B P— 5 7 8 8 ) の斜面培養から一白 金耳 、 1 0 0 m lの液体培地 〔ディ フコイース トナイ トロジヱンベー ス 0. 6 7 % (wZv) 、 グルコース 2. 0 % ( w/ V ) 〕 を入れた 5 0 0 m l容の三角フラスコに接種し、 2 5 °Cで 5 日間振 ¾し、 種培養 液を得た。 この種培養液 1. 0 m 1を、 上記液体培地 1 2 5 m l を入れ た 5 0 0 m 1容の三角フラスコ 1 8本に接種し、 2 5 °C、 9日間振盪培 養 （振通 2 2 0 r pm) を行った。 このようにして得た培養液を遠心分 離し、 上澄み液と菌体とに分離した。 得られた菌体にメ タノ ール 1 Lを 加えて充分混合して抽出操作をした後、 '减圧濃縮を行った。 得られた残 渣に水を 3 0 0 m l加えて充分混合した後、 p Hを 2に調整した。 これ に酢酸ェチルを 3 0 0 m l加えて充分混合し、 酢酸ェチル洗浄操作を行 つた。 この水層の p Hを 9に調整し、 酢酸ェチルを 3 0 0 m 1加えて抽 出操作を行った。 この抽出液を减圧濃縮し、 残淹 5 2 m gを得た。  From the slant culture of the TKR 1785 strain (FE RM BP-5788), 100 ml of liquid medium, 100 ml of liquid medium (Difcoyce Tony Tradion base 0.67% (wZv), glucose 2. 0% (w / V)] was inoculated into a 500 ml Erlenmeyer flask, and shaken at 25 ° C for 5 days to obtain a seed culture. 1.0 ml of this seed culture was inoculated into 18 500 ml Erlenmeyer flasks each containing 125 ml of the above liquid medium, and cultured at 25 ° C for 9 days with shaking. 220 rpm). The culture solution thus obtained was centrifuged and separated into a supernatant and cells. To the obtained cells, 1 L of methanol was added, mixed well, and extracted, and then concentrated under reduced pressure. After adding 300 ml of water to the obtained residue and mixing well, the pH was adjusted to 2. To this, 300 ml of ethyl acetate was added and mixed well, followed by washing with ethyl acetate. The pH of this aqueous layer was adjusted to 9, and 300 ml of ethyl acetate was added to perform extraction. The extract was concentrated under reduced pressure to obtain 52 mg of residual brew.
得られた残渣をメ タノ ール 0. 4 m l に溶解し、 高速液体クロマ トグ ラフィ一に付し、 2つの抗真菌活性画分 I及び I Iを得た。 それぞれの 画分を減圧濃縮することにより、 TKR 1 7 8 5— I及び TKR 1 7 8 5 — I Iの精製物 1 6 m g及び 3 mgを白色粉末として得た。 なお、 高 速液体ク口マ トグラフィ一の条件は下記によつた。  The obtained residue was dissolved in 0.4 ml of methanol and subjected to high performance liquid chromatography to obtain two antifungal active fractions I and II. The fractions were concentrated under reduced pressure to obtain 16 mg and 3 mg of purified TKR1785-I and TKR1785-II, as white powders. The conditions for high-speed liquid mouth chromatography were as follows.
装置 ： L C 8 A (島津製作所社製）  Equipment: L C 8 A (manufactured by Shimadzu Corporation)
カラム ： YMC p a c k C , β ( 2. 0 c m x 2 5 c m) (ヮイエム I 6 Column: YMC pack C, β (2.0 cm x 25 cm) I 6
シ一社製） Made by Shisha)
移動相 ： 0. 0 5 % ト リ フルォロ酢酸を含む 5 5 % ( V / V ) ァセ 卜 二 ト リル/水 理化学的性質  Mobile phase: 5.5% (V / V) acetate containing 0.05% trifluoroacetic acid / Hydrochemical properties
質量分析には、 JMS— D X 3 0 2型質量分析装置 （日本電子社製） を用いた。 'H— NMRスぺク トル （重ジメチルスルホキン ド中、 標準 物質 ： 重ジメチルスルホキシ ド） 及び13 C一 NMRスぺク トル （重ジメ チルスルホキシ ド中、 標準物質 ： 重ジメチルスルホキシ ド） の測定には、 J NM- A 5 0 0核磁気共鳴装置 （日本電子社製） を用いた。 紫外線吸 収スぺク トル分析 （メ タノール中） には、 UV— 2 5 0型自記分光光度 計 （島津製作所社製） を用いた。 赤外線吸収スぺク トル分析 （KB r法） には、 2 7 0— 3 0型赤外分光光度計 （日立製作所社製） を用いた。 ァ ミ ノ酸分析には、 L— 8 5 0 0型 （日立製作所社製） を用いた。 以下に TKR 1 7 8 5 - 1の理化学的性質を述べる。 A JMS-DX302 mass spectrometer (manufactured by JEOL Ltd.) was used for mass spectrometry. 'H-NMR spectrum (in heavy dimethyl sulfoxide, standard substance: heavy dimethyl sulfoxide) and 13C- NMR spectrum (in heavy dimethyl sulfoxide, standard substance: heavy dimethyl sulfoxide) Was measured using a JNM-A500 nuclear magnetic resonance apparatus (manufactured by JEOL Ltd.). For UV absorption spectrum analysis (in methanol), a UV-250 type recording spectrophotometer (manufactured by Shimadzu Corporation) was used. For infrared absorption spectrum analysis (KBr method), a 270-30 type infrared spectrophotometer (manufactured by Hitachi, Ltd.) was used. For amino acid analysis, L-850 type (manufactured by Hitachi, Ltd.) was used. The physicochemical properties of TKR 1785-1 are described below.
高速液体ク口マ トグラフィ一に付し、 得られた活性画分 Iを減圧澳縮 することにより得られた白色粉末精製物は、 質量分析による F A B— M S測定で、 mZz 5 1 8 [M+H] + であることが判明した。 本物質に ついての 'Η— NMRスぺク トル測定及び：3C— NMRスぺク トル測定 並びにその解析により、 炭素数 2 7であり、 窒素数 3であることが判つ た。 1 H— NMRスペク トルを図 3に、 ： 3 C— NMRスペク トルを図 4 に示した。 更に、 本物質のメタノール中における紫外線吸収スペク トル は、 図 1に示すように、 末端吸収を示すことが判った。 本物質の K B r 法による赤外線スぺク トル測定結果は、 下記のとおりであった。 赤外線 吸収スぺク トルを図 2に示した。 I R (K B r ) (cm —1 ) : 3 4 1 0、 2 9 2 0、 2 8 5 0、 1 6 7 0 1 5 4 0、 1 4 7 0、 1 2 1 0 、 1 1 4 0、 1 0 5 0 、 8 4 0 , 8 0 0 、 7 2 0 o The purified white powder obtained by subjecting the obtained active fraction I to a high-pressure liquid chromatography and reducing the pressure under reduced pressure was analyzed by FAB-MS by mass spectrometry to obtain mZz 518 [M + H] + . According to 'Η-NMR spectrum measurement and 3 C-NMR spectrum measurement of this substance and analysis thereof, it was found to have 27 carbon atoms and 3 nitrogen atoms. The 1 H-NMR spectrum is shown in FIG. 3, and the: 3 C-NMR spectrum is shown in FIG. Furthermore, it was found that the ultraviolet absorption spectrum of this substance in methanol showed terminal absorption as shown in FIG. The infrared spectrum measurement result of this substance by the KBr method was as follows. Figure 2 shows the infrared absorption spectrum. IR (KB r) (cm — 1 ): 3 4 1 0, 2 9 2 0, 2 8 5 0, 1 6 7 0 1 5 4 0, 1 4 7 0, 1 2 1 0, 1 1 4 0, 1 0 5 0, 8 4 0, 8 0 0, 7 2 0 o
また、 本物質の各種溶媒に対する溶解性は、 メ タノール、 水に可溶、 クロ口ホルム、 へキサンには難溶であった。  The solubility of this substance in various solvents was soluble in methanol and water, and slightly soluble in chloroform and hexane.
上記分析結果により、 高速液体クロマ トグラフィ ーに付し、 得られた 活性画分 Iを減圧鏖縮することにより得られた白色粉末精製物は、 TK 尺 1 7 8 5— 1でぁることが判明した。 また、 図 3の 'H— NMRスぺ ク トル及び図 4の'3 C— NMRスぺク トルについて詳細に解析したとこ ろ、 TKR 1 7 8 5— Iは、 式 （I) の化学構造を有することが明らか となった。 According to the above analysis results, a purified white powder obtained by subjecting the obtained active fraction I to high-performance liquid chromatography and subjecting the obtained active fraction I to reduced pressure evacuation can be obtained using a TK scale 1785-1. found. Further, detailed analysis of the 'H-NMR spectrum of FIG. 3 and the' 3 C-NMR spectrum of FIG. 4 shows that TKR1785-I has the chemical structure of the formula (I) It became clear that it had.
TKR 1 7 8 5— Iを LC— 1 0 A型高速液体クロマ 卜グラフィー装 置 （島津製作所社製） を用いた逆相分配高速液体ク口マ トグラフィ 一 (HPLC) による分析に供した。 なお、 高速液体クロマ トグラフィー の条件は下記によつた。  TKR1785-I was subjected to analysis by reversed-phase partition high-performance liquid chromatography (HPLC) using an LC-10A high-performance liquid chromatograph (manufactured by Shimadzu Corporation). The conditions for high performance liquid chromatography were as follows.
カラム ： CAPCEL L PACK C , « ( 6 mm x 1 5 0 m m) (资 生堂社製） Column: CAPCEL L PACK C, «(6 mm x 150 mm) () manufactured by Iseido Co., Ltd.)
移動相 ： 0. 0 5 %ト リ フルォロ酢酸を含む 5 0 % ( v/v) ァセ ト 二 ト リルノ水  Mobile phase: 0.05% (v / v) aqueous solution containing 5% trifluoroacetic acid
カラム温度 ： 4 0。C  Column temperature: 40. C
検出 U V波長 ： 2 2 0 nm  Detection UV wavelength: 220 nm
分析の結果、 TKR 1 7 8 5— Iは、 図 5に示す位置に溶出されるこ とが明らかとなった。 次に、 TKR 1 7 8 5 - 1 1の理化学的性質を述べる。  As a result of the analysis, it was revealed that TKR1785-I was eluted at the position shown in FIG. Next, the physicochemical properties of TKR 1785-11-1 are described.
高速液体ク口マ トグラフィ一に付し、 得られた活性画分 I Iを減圧濃 縮することにより得られた白色粉末精製物について、 理化学的性質を調 ベた。 本物質は、 質量分析による F A B— M S測定で、 mZz 5 3 2 [M— H: - であり、 TKR 1 7 8 5— Iより 1 4マス分子量が大きい ことが判明した。 また、 塩酸加水分解後、 ア ミ ノ酸分析にかけたところ、 T K R 1 7 8 5— Iは L一バリ ンを含有しているのに対し、 本物質は、 L—イソロイシンを含有していることが明らかとなった。 紫外線吸収ス ベタ トルは TKR 1 7 8 5 - 1とほとんど差がなかった。 また、 本物質 の各種溶媒に対する溶解性も、 TKR 1 7 8 5 - 1と類似していた。 本 物質の 'Η— NMRスぺク トル （図 6) 、 '3C— NMRスぺク トル （図 7 ) の結果を合わせて検討した結果、 本物質は、 式 （ I I ) の化学構造 を有することが明らかとなった。 High-performance liquid chromatography was performed, and the obtained active fraction II was concentrated under reduced pressure. The physicochemical properties of the purified white powder obtained by compression were examined. The substance was found to be mZz532 [M-H:-by FAB-MS measurement by mass spectrometry, and was found to be 14 mass molecular weights greater than TKR1785-I. In addition, analysis of amino acid after hydrolysis of hydrochloric acid revealed that TKR1785-I contained L-valin, whereas this substance contained L-isoleucine. Became clear. The UV absorption spectra were almost the same as TKR 1785-1. The solubility of this substance in various solvents was similar to that of TKR1785-1. Of the material '.eta. NMR spectrum (FIG. 6),' 3 C-NMR spectra results of investigation combined results (Figure 7), the substance, the chemical structure of formula (II) It became clear to have.
上記分析結果により、 高速液体クロマ トグラフ ィーに付し、 得られた 活性画分 I Iを減圧濃縮することにより ί晷られた白色粉末精製物は、 Τ KR 1 7 8 5 - I Iであることが判明した。  According to the above analysis results, the purified white powder obtained by high-performance liquid chromatography and concentrating the obtained active fraction II under reduced pressure was KR 1785-II. found.
TKR 1 7 8 5— I Iを L C一 1 0 Α型高速液体ク口マ トグラフィー 装置 （島津製作所社製） を用いた HPL Cによる分析に供した。 なお、 高速液体ク口マ トグラフィ一の条件は、 上記 TKR 1 7 8 5 - 1を分析 した場合と同一であった。 分析の結果、 上記 TKR 1 7 8 5— I Iは、 図 8に示す位置に溶出されることが明らかとなつた。 生物学的性質  TKR1785-II was subjected to analysis by HPLC using an LC-10Α type high-performance liquid chromatograph (manufactured by Shimadzu Corporation). The conditions for high performance liquid mouth chromatography were the same as in the above analysis of TKR1785-1. As a result of the analysis, it was revealed that the above TKR1785-II was eluted at the position shown in FIG. Biological properties
( 1 ) 抗真菌活性  (1) Antifungal activity
得られた TKR 1 7 8 5類を使用して各種微生物に対する抗菌スぺク トルを調べた。 測定は、 液体培地希釈法により、 菌の増殖をほぼ完全に 阻止した濃度を最小生育阻害澳度 （ gZm 1 ) と して求めた。 結果を 表 2に示した。 また、 部分的に菌の增殖を阻害する最小濃度を半阻止濃 度 （/ gZm 1 ) として求め、 併せて表 2の括弧内に示した。 表中、 Y N B Gは、 イース トナイ トロジェンベース （ディ フ コ社製） 0. 6 7 %. グルコース 1 %を含有する培地を表す。 BH Iは、 ブレイ ンハー トイ ン ヒユージョ ンブイヨン （曰水製薬社製） 0. 5 %を含有する培地を表す: YN B G— Tw e e nは、 イース トナイ トロジェンベース 0. 6 7 %、 ノくク トカシ ト ン 0. 5 %、 グルコース 2 %、 ツイ 一ン 4 0. 1 %を含有 する培地を表す。 表 2 The antibacterial spectrum against various microorganisms was examined using the obtained TKR1785. The concentration was determined as the minimum growth inhibition level (gZm 1) by the liquid medium dilution method, at which the growth of the bacteria was almost completely inhibited. Table 2 shows the results. In addition, the minimum concentration that partially inhibits bacterial growth is The degree (/ gZm 1) was obtained and is shown in parentheses in Table 2. In the table, YNBG indicates a medium containing 0.67% of yeast tonite trogen base (manufactured by Difco) and 1% of glucose. BHI stands for a medium containing 0.5% of Brainhard Toin Hydrange Bouillon (manufactured by Mizusui Pharmaceutical Co., Ltd.): YN BG—Tween is 0.67% of yeast nutrient base, Represents a medium containing 0.5% of ton, 2% of glucose and 40.1% of tween. Table 2
Figure imgf000021_0001
Figure imgf000021_0001
—— . ：制定せず  ——.: Not established
表 2の結果から、 本発明の生理活性物質 T KR 1 7 8 5類は、 カ ンジ ダ · アルビカ ンス、 カ ンジダ ' ケフィ ール、 ク リプ ト コ ッカス ' ネオホ ルマンス、 マラセチア ' ファーファー等の病原性真菌に対して抗菌活性 を有することが判明した。 ( 2 ) 各種酵素阻害活性 From the results in Table 2, it can be seen that the physiologically active substances TKR1785 of the present invention include Candida albicans, Candida 'Kefir', Cryptococcus 'Neoholmans, Malassezia' and Farfer. Antibacterial activity against pathogenic fungi Was found to have. (2) Various enzyme inhibitory activities
TKR 1 7 8 5 — I について、 免疫系に関与する酵素であるホスホリ パーゼ A 2 (ブタ睇臓由来） 及びロイコ ト リェン （L T) 合成酵素For TKR 1 785-I, enzymes involved in the immune system, phospholipase A 2 (derived from swine spleen) and leukotriene (LT) synthase
(モルモッ 卜肺由来） に対する阻害活性を測定した。 ホスホリパーゼ As 活性は、 [ C] ホスファチジルコリ ンから生成する [" C] パルミ チ ン酸量を酸性下へキサン抽出物について測定した。 また、 L T C , 合成 酵素活性は、 LTA, から生成する L T C , の量を R I A法で測定した。 その結果を表 3に示した。 表 3 (Derived from guinea pig lung) was measured. Phospholipase As activity was determined by measuring the amount of ["C] palmitic acid produced from [C] phosphatidylcholine in acidic hexane extracts. In addition, the activity of LTC and synthase was measured by the activity of LTC and Was measured by the RIA method, and the results are shown in Table 3. Table 3
Figure imgf000022_0001
Figure imgf000022_0001
TKR 1 7 8 5 — ： [ は、 ァ レルギ一反応に関与する酵素であるホスホ リパーゼ A2 及び L T C < 合成酵素を阻害した。 TKR 1 7 8 5 -: [inhibited phospholipase A 2 and LTC <synthase, an enzyme involved in § Rerugi first reaction.
( 3 ) 混合リ ンパ球反応 （ML R) 阻害活性 C 5 7 B LZ 6マウス及び B A L B/ cマウスより脾臓を摘出し、 培 地中でホモゲナイズし、 細胞懸濁液とした。 C 5 7 B LZ 6由来の細胞 懸濁液をナイ口 ンウールカラムにかけ、 T細胞リ ッチなものを調製した (レスポンダー細胞） 。 B A L B/c由来の細胞懸濁液を X線照射し、 スティ ムレ一タ一細胞を調製した。 レスポンダー細胞とステイ ムレ一タ 一細胞を 1 ： 1の割合で混ぜ、 C O 2. イ ンキュベータ—内で、 4 日間培 養後、 H—チミ ジンを加え、 更に一晩培養後、 細胞を回収し、 3H— チ ミ ジン取り込み量を測定した。 サンプル （ジメチルスルホキン ド溶液 を培地で希釈した 5 0 0、 1 2 5、 3 1. 2、 7. 8 g/m 1溶液） は、 レスポンダー細胞とスティムレーター細胞とを混ぜる際に 0. 5 % 加え、 Φ終濃度 2 5〜0. 0 3 9 g/m 1 とした。 サンプル無添加群 の 3H—チミ ジン取り込み量と比較して、 阻害活性を求めた。 TK R 1 7 8 5 - 1は濃度依存的な] VILR阻害活性を示し、 その 5 0 %阻害'港 度は、 0. 4 1 /i g/m 1であった。 (3) Mixed lymphocyte reaction (MLR) inhibitory activity The spleen was excised from the C57BLZ6 mouse and BALB / c mouse, and homogenized in the medium to obtain a cell suspension. A cell suspension derived from C57B LZ6 was applied to a nylon wool column to prepare a T cell-rich cell (responder cells). A cell suspension derived from BALB / c was irradiated with X-rays to prepare stimulator cells. Responder cells and stimulator cells are mixed at a 1: 1 ratio, cultured in a CO 2 .incubator for 4 days, H-thymidine is added, and after overnight culture, the cells are collected. And 3 H-thymidine incorporation were measured. Samples (500, 125, 31.2, and 7.8 g / ml solutions of dimethylsulfoxide solution diluted in culture medium) were used when mixing the responder cells with the stimulator cells. % In addition, the Φ final concentration was set to 25 to 0.039 g / m 1. The inhibitory activity was determined by comparing the amount of 3 H-thymidine incorporation in the sample-free group. TK R 1785-1 shows a concentration-dependent] VILR inhibitory activity, and its 50% inhibitory activity was 0.41 / ig / m1.
すなわち、 ァレルギ一を始めとする免疫反応を阻害する効果があるこ とが明らかとなった。  In other words, it has been clarified that there is an effect of inhibiting allergic reactions and other immune reactions.
( 4 ) 毒性 (4) Toxicity
得られた T K R 1 7 8 5— I及び T K R 1 7 8 5 - I Iを、 それぞれ I C R系マウスに 5 0 m g/k gを腹腔内投与したが、 毒性は認められ 力、つた。 The obtained TKR1785-I and TKR1785-II were intraperitoneally administered to ICR mice at 50 mg / kg, respectively.
製剤例 1 Formulation Example 1
T K R 1 7 8 5 - I 5 0 m g  T K R 1 7 8 5-I 50 mg
乳糖 4 6 m g  Lactose 4 6 mg
トウモロコ シデンプン 2 0 m g  Corn starch 20 mg
低置換度ヒ ドロキシプロビルセルロース 8 m g  Low substituted hydroxypropyl cellulose 8 mg
ヒ ドロキンプロ ビルメチルセルロース 5 m g  Hydroquinprovir methylcellulose 5 mg
ステアリ ン酸マグネシゥム 1 m g  Magnesium stearate 1 mg
叶 1 3 0 m g  Kano 1 3 0 mg
ヒ ドロキシブ口ピルメチルセルロース及びステアリ ン酸マグネシゥム を除く上記処方成分を均一に混合した後、 ヒ ドロキンプロピルメチルセ ルロース 8 % (w/w) 水溶液を結合剤として湿式造粒法にて打錠用顆 粒を製造した。 これにステアリ ン酸マグネシウムを混合した後、 打錠機 を用いて直径 7 mm、 1錠重量 1 3 0 m gに形成し、 内服錠とした。 製剤例 2  After uniformly mixing the above-mentioned ingredients except for pillmethylcellulose and magnesium stearate, the tablets are made by wet granulation using a 8% (w / w) aqueous solution of hydroxyquinpropylmethylcellulose as a binder. Condyles were produced. After mixing with magnesium stearate, it was formed into a tablet with a diameter of 7 mm and a weight of 130 mg per tablet using a tableting machine. Formulation Example 2
TKR 1 7 8 5 - I 1 S  TKR 1 7 8 5-I 1 S
吸水軟膏 （日本薬局方収載） 9 9 g  Water-absorbing ointment (listed in the Japanese Pharmacopoeia) 9 9 g
計 1 0 0 g  Total 100 g
TKR 1 7 8 5 - I をまず少量の吸水软青と充分に練り合わせ、 つい で、 残った吸水軟青を徐々に加えて均一になるまで練り合わせて钦裔を 作製した。 得られた软音は、 1 日 4〜 5回患部に塗布されるものであつ た。 産業上の利用可能性  First, TKR 1785-I was kneaded sufficiently with a small amount of water-absorbing blue, and then the remaining water-absorbing soft blue was gradually added and kneaded until uniform to produce descendants. The resulting sound was applied to the affected area 4 to 5 times a day. Industrial applicability
本発明により、 真菌感染症、 アレルギー性疾患及び免疫の治療剤等の 臨床医薬として有用である生理活性物質 TKR 1 7 8 5類及びその製造 方法を提供することができる。 INDUSTRIAL APPLICABILITY According to the present invention, a physiologically active substance TKR1785 useful as a clinical drug such as a therapeutic agent for fungal infections, allergic diseases and immunity, and the production thereof A method can be provided.

Claims

請求の範囲 The scope of the claims
1. 下記一般式 （ A) ： 1. The following general formula (A):
OH OH R CH20H OH OH R CH 2 0H
I I I  I I I
H3C-(CH2)i2-CH-CH2-CH-CH— CH2-CO-NH-CH-CO-NH-CH-CH2OH H 3 C- (CH 2 ) i 2 -CH-CH2-CH-CH— CH2-CO-NH-CH-CO-NH-CH-CH2OH
NH2 NH 2
(A)  (A)
(式中、 Rは、 — CH (C H 3 ) 2 又は一 CH (CH. ) C 2 H 0 であ る。 ） で表される生理活性物質 TKR 1 7 8 5類。 (Wherein, R is —CH (CH 3) 2 or one CH (CH.) C 2 H 0).
2. ぺニシリウム (P e n i c i 1 1 i urn) 厲に厲する菌株であって、 生理活性物質 TKR 1 7 8 5類を産生する菌株を培養し、 その後、 前記 菌株の培養物から目的物を単離することを特徴とする生理活性物質 TK R 1 7 8 5類の製造方法。 2. Culture of a strain of Penicillium (Penici 11 i urn), which produces the physiologically active substance TKR1785, and then isolate the target substance from the culture of the strain. A method for producing a physiologically active substance TK R 1 785, characterized in that it is released.
3. ベニシリウム (P e n i c i I l i um) 厲に厲し、 生理活性物質 TKR 1 7 8 5類を産生することを特徴とする微生物。 3. Benicillium (PenicIlium) A microorganism characterized by producing the physiologically active substances TKR1785.
4. 生理活性物質 TKR 1 7 8 5類を含有することを特徴とする医薬組 成物。 4. A pharmaceutical composition characterized by containing a physiologically active substance TKR1785.
5. 医薬組成物が、 生理活性物質 TKR 1 7 8 5類を有効成分とする抗 ァレルギ一剤である請求項 4記載の医薬組成物。 5. The pharmaceutical composition according to claim 4, wherein the pharmaceutical composition is an antiallergic agent containing a physiologically active substance TKR1785 as an active ingredient.
6. 医薬組成物が、 生理活性物質 TKR 1 7 8 5類を有効成分とする免 疫調節剤である請求項 4記載の医薬組成物。 6. The pharmaceutical composition is exempt from the use of the biologically active substance TKR 178 5. The pharmaceutical composition according to claim 4, which is a disease regulator.
PCT/JP1997/000770 1996-03-12 1997-03-12 Physiologically active substances tkr1785's, process for the preparation thereof, and microbe WO1997034012A1 (en)

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EP97907278A EP0897988B1 (en) 1996-03-12 1997-03-12 Physiologically active substances tkr1785's, process for the preparation thereof, and microbe
JP53243897A JP3273948B2 (en) 1996-03-12 1997-03-12 Physiologically active substances TKR1785s, production method and microorganism
DE69733016T DE69733016T2 (en) 1996-03-12 1997-03-12 PHYSIOLOGICALLY ACTIVE TKR1785'S SUBSTANCES, PROCESS FOR THEIR PREPARATION AND MICROBE

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999012890A1 (en) * 1997-09-11 1999-03-18 Takara Shuzo Co., Ltd. Sphingosine derivatives and medicinal composition
WO1999032498A1 (en) * 1997-12-22 1999-07-01 Takara Shuzo Co., Ltd. Antibiotic tkr2999, process for the preparation thereof and microbe

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KR100559817B1 (en) 1997-03-12 2006-03-10 다카라 바이오 가부시키가이샤 Sphingosine analogues
AU2012316085A1 (en) * 2011-09-29 2014-05-22 Emory University Sphingosine analogs, compositions, and methods related thereto

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4554289A (en) * 1982-12-25 1985-11-19 Takara Shuzo Co., Ltd. Of4949

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
US4554289A (en) * 1982-12-25 1985-11-19 Takara Shuzo Co., Ltd. Of4949

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP0897988A4 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999012890A1 (en) * 1997-09-11 1999-03-18 Takara Shuzo Co., Ltd. Sphingosine derivatives and medicinal composition
WO1999032498A1 (en) * 1997-12-22 1999-07-01 Takara Shuzo Co., Ltd. Antibiotic tkr2999, process for the preparation thereof and microbe
US6333305B1 (en) 1997-12-22 2001-12-25 Takara Shuzo Co., Ltd. Antibiotic TKR2999, process for the preparation thereof and microbe

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US6103767A (en) 2000-08-15
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